Cellular tasks are accomplished by specialized macromolecular machines. The operating principles of most of these machines have been elucidated in great detail, especially since the advent of single-molecule techniques. However, much less attention has been given to the mechanism of coordination between different macromolecules, which occurs frequently inside the cell. The candidate's long-term goal is to understand how essential cellular machines, especially the ones that are closely related to human diseases, interact with one another and how they act in concert in response to environmental changes. In this proposal, the candidate plans to study the interactions between three key bacterial enzymes, namely RNA polymerase (RNAP), the ribosome, and transcription termination factor Rho. In the first specific aim, the candidate will develop an in vitro transcription-translation coupling system tailored for a single-molecule optical tweezers assay. This assay will monitor in real time the transcriptional dynamics of E. coli RNAP in the presence of a translating ribosome, which makes it possible to quantitatively understand the crosstalk between two principle gene expression machineries. This assay will then be used as a platform to characterize the dynamics of transcription- translation coupling under various cell growth conditions, and to evaluate the roles of protein cofactors and small-molecule inhibitors in the coupling. In the second aim, the candidate will investigate the molecular mechanism by which Rho, a universal transcription termination factor found in most bacteria, interacts with RNAP and terminates RNA synthesis. He will use high-resolution optical tweezers to characterize the mechanochemical properties of Rho as a motor protein. With this knowledge, he will further probe the interaction between the translocating Rho and the transcribing RNAP by direct observation at the single- molecule level. Finally, after understanding the mechanism of coupling between RNAP and the ribosome, and between RNAP and Rho, the candidate wishes to explore the concerted action of all three enzymes, which has been postulated to play a crucial role in the regulation of bacterial gene expression. Therefore elucidating the cooperative mechanism among these molecular machines may offer new opportunities for antibiotic development. The mentored phase of the project will be conducted in Dr. Carlos Bustamante's laboratory at UC Berkeley. Dr. Ignacio Tinoco will serve as the candidate's co-mentor. The research will benefit from the state- of-the-art facilities and resources at QB3-Berkeley Institute and the expertise of the Bustamante Lab and the Tinoco Lab in single-molecule transcription and translation studies. The key area in which the candidate wishes to acquire additional training is the advanced molecular biology and biochemistry skills necessary for developing the single-molecule transcription-translation coupling system. The candidate also plans to utilize the mentored phase of the award period to enhance his skills in lab management, teaching, scientific communication, and grant writing. These skills will facilitate his successful transitionto independence.